Determining the chemical composition of complex biological systems, such as tissues, biofilms, and microbial colonies, presents a daunting analytical challenge. The compositions of such samples are typically heterogeneous and dynamic, changing both in time and in response to varying environmental conditions. This necessitates the use of methods of analysis that can provide chemical information with both high spatial and temporal resolution. The ability to measure and image the chemical composition of biological samples under native conditions and with minimal modification/preparation is important to advancing the understanding of processes, such as cell differentiation, photosynthesis, and cellular metabolism during stress-and-adaptive response, and nonenzymatic glycation in cardiac tissues induced by a high glycemic index diet.
There are many microanalysis techniques for characterizing the chemical composition of biological samples, including NMR/MRI, visible microscopy, infrared spectromicroscopy, Raman imaging, fluorescence-tagging and imaging of molecules, and imaging mass spectrometry. Many of these techniques can provide high spatial resolution and are nondestructive, but often do not provide unambiguous chemical information. Fluorescence-tagging of molecules can be used to obtain images with both high spatial resolution (˜1-200 nm) and high molecular specificity by using antibodies to target specific molecules. However, only a few components can be imaged simultaneously, and the procedure for tagging molecules with fluorophores often requires extensive sample preparation. Imaging mass spectrometry provides chemical information with excellent molecular specificity, and thousands of compounds can be measured simultaneously. Mass spectrometry can also be combined with other imaging techniques to provide multimodal imaging analysis. Unlike many optical methods, mass spectrometry is a destructive technique; molecules must be removed from the sample and ionized to be detected.
The most widely used mass spectrometry imaging techniques are matrix-assisted laser desorption ionization (MALDI) and secondary ion mass spectrometry (SIMS). Conventional MALDI and SIMS are often used to generate chemical images for fixed tissue samples. For both techniques, ions are generated in vacuum and are subsequently mass analyzed. Because vacuum is required for these techniques, neither is suitable for the analysis of living systems. MALDI typically involves the application of an external and usually denaturing matrix molecule which absorbs the energy from a laser for ablation and ionization. With SIMS, secondary ions are sputtered from a surface with a beam of primary ions, such as Cs+ or polyatomic Aun+ clusters. Chemical images can be obtained with very high spatial resolution (˜100 nm), but the sensitivity for high mass ions (m/z>1000) is often low.
Many techniques for imaging mass spectrometry at ambient pressure have been recently introduced. Cooks and co-workers developed a now widely used method, desorption electrospray ionization (DESI), in 2004. With DESI, charged solvent droplets generated by electrospray are directed toward a sample surface to desorb and ionize the sample at the surface. Many other ambient imaging mass spectrometry techniques have subsequently been developed. With nano-DESI and liquid microjunction surface sampling probe (LMJ-SSP), the solvent contacts a small area of a sample and is then directed to an ESI source. Numerous methods use laser light to select spatially resolved areas for mass analysis. These methods include atmospheric pressure infrared MALDI (AP IR-MALDI), electrospray-assisted laser desorption ionization (ELDI), matrix-assisted laser desorption electrospray ionization (MALDESI), laser ablation electrospray ionization (LAESI), laser ablation capillary electrophoresis electrospray ionization (CE-ESI), and IR, visible light, or near-IR laser ablation sample transfer (LAST).
Methods that use IR-laser ablation can take advantage of the water naturally present in biological samples as a matrix to absorb IR radiation. The IR laser pulse produces surface evaporation, phase explosion (explosive boiling) of water, and the secondary ejection of sample material into a plume of fine droplets. The ejected sample material consists of mostly neutral droplets, particles, or molecules, which can be ionized by intersection with an electrospray plume (ELDI, LAESI, and MALDESI), or can be captured in solvent through LAST for subsequent ionization by electrospray, or for capillary electrophoresis separation prior to subsequent ionization by electrospray. The fraction of ions that are produced directly can be introduced into the mass spectrometer, for example atmospheric pressure infrared MALDI (AP IR-MALDI). Little excess energy is deposited into solute molecules in IR laser ablation. Studies with thermometer ions and peptides show that LAESI produces ions with internal energies indistinguishable from those produced by ESI, indicating that the laser ablation process itself is soft.
High transfer efficiency of the sample to the mass spectrometer is especially important for the analysis of biological samples due to low concentrations of some molecular species within the highly complex mixtures of biochemicals from living cells. The transfer efficiency of laser ablated material from a surface to a solvent probe depends on the instrumental geometry. In experiments where backside laser ablation was used, the transfer efficiency for ablated angiotensin II in solution on a quartz slide to solution on a probe 1 mm away is reported to be 2%. LAESI, in which the ablation plume expands into a flow of highly charged solvent droplets produced by electrospray, is reported to be “characterized by significant sample losses and low ionization efficiencies.” Vertes and co-workers reported that the transfer efficiency of LAESI was improved by the use of a capillary to confine the sample and to direct the radial expansion of the ablation plume, guiding more material directly into the electrospray flow.
Details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims. Note that the relative dimensions of the following figures may not be drawn to scale.